Abstract
We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.
🔬 Techniques
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Figure 1
Reversible quenching of Cy5 by TCEP. (A) Absorption spectrum of Cy5 incubated with various concentrations of TCEP, showing a decrease in absorption at 645 nm and an increase in absorption at ~350 nm. ...
Figure 2
1 H NMR spectrum at 21 u00b0C (600 MHz, D 2 O) of the polymethine bridge protons for the di-deuterio Cy5-dTCEP adduct. Two-, three-, and four-bond phosphorus-hydrogen coupling was observed in the doub...
Figure 3
Two-color STORM image of immunolabeled microtubules in a BS-C-1 cell. (A) Tyrosinated tubulin was stained with Alexa 647 (magenta) and detyrosinated tubulin was stained with Alexa 750 (green). (Bu2013...
Figure 4
Internalization of Alexa 647-labeled transferrin in A549 cells measured by confocal microscopy. (A) Surface-bound transferrin was quenched upon addition of TCEP to the imaging medium, but (B) internal...
Scheme 1
Formation and structure of Cy5-TCEP adduct.
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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